γ-Secretase processes a variety of substrates including the amyloid precursor protein (APP) and Notch proteins. γ-Secretase cleaves APP to release Aβ peptides, which are widely considered to play a causative role in Alzheimer disease (AD).
The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms (Artavanis-Tsakonas et al., 1999, Science 284 (5415): 770-776). Notch protein family members are present in all metazoans, and mammals possess four different Notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. The Notch receptor is a single-pass transmembrane receptor protein. The ligands that bind Notch proteins and initiate signaling are bound to neighboring cells. Notch is a hetero-oligomer composed of a large extracellular portion, which associates in a calcium-dependent, non-covalent interaction with a smaller piece of the Notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region (Brou et al., 2000, Mol. Cell. 5 (2): 207-16).
In addition to acting on APP, γ-secretase processes Notch and other type I membrane proteins. Notch is also cleaved by multiple proteases. Notch signaling is controlled by ligand binding, which exposes a negative control region that is susceptible to proteolytic cleavage of the receptor. The GenBank Accession Number for human Notch1 cDNA is AF'308602.1, and for the corresponding polypeptide gene product it is AAG33848.1. Human Notch1 has 2556 amino acid residues. In processing Notch1, first, a membrane-proximal cleavage is executed at site S2 (between residues Ala1710 and Val1711 (see Supplementary Figure S1A of van Tetering et al., 2009, J. Biol. Chem., 284:31018-31027) by an ADAM (A Disintegrin And Metalloprotease) metalloprotease, removing the extracellular domain to generate the membrane-anchored Notch extracellular truncation fragment (NEXT). (The ADAM family includes proteins containing disintegrin-like and metalloprotease-like domains. ADAMs are involved in diverse cellular processes such as development, cell-cell interactions and protein ectodomain.) NEXT is subsequently cleaved by γ-secretase at site S3 (see Supplementary Figure S1A of van Tetering et al., 2009, J. Biol. Chem., 284:31018-31027) to generate Notch Intracellular Domain (NICD) which translocates into the nucleus where it modulates expression of target genes that are involved in cell fate decisions during embryogenesis, hematopoeisis, and stem cell differentiation (Chan et al., 1998, Cell, 94, 423-426, Berezovska et al., 1999, Brain Res Mol Brain Res., 69:273-280, and Redmond et al., 2000, Nat. Neurosci., 3:30-40).
γ-Secretase is a membrane-bound aspartyl protease consisting of at least four subunits, which are Presenilin (PS, either PSlor PS2), Aph-1, Nicastrin (Nct) and Pen-2. PS is believed to be the catalytic subunit of γ-secretase and mutations in PS1 and PS2 have been linked to Familial Alzheimer's Disease (FAD) (Sherrington et al., 1995, Nature, 375:754-760, Levy Lahad et al., 1995, Science, 269:973-977). Although the precise pathological mechanism of these FAD mutations is unknown, it has been postulated that they alter the specificity of γ-secretase and lead to an increase in the ratio of Aβ42 to Aβ40 peptide (Borchelt et al., 1996, Neuron, 17:1005-1013, Duff et al., 1996, Nature, 383:710-713, and Bentahir et al., 2006, J. Neurochem., 96:732-742) Aβ42 is more prone to form insoluble aggregates than is Aβ40, so that FAD mutations are deleterious. In addition, FAD mutations also reduce γ-secretase activity for Notch and E-cadherin processing (Song et al., 1999, Proc. Nat. Acad. Sci. USA, 96:6959-6963, Marambaud et al., 2002, EMBO J., 21:1948-1956, and Marambaud et al., 2003, Cell, 114:635-645). Several studies have shown that FAD mutations of PS1 affect Notch cleavage (Bentahir et al., 2006, J. Neurochem., 96:732-742), Song et al., 1999, Proc. Nat. Acad. Sci. USA, 96:6959-6963, Nakajima et al., 2000, J Neurosci Res., 62:311-317, Amtul et al., 2002, Neurobiol Dis., 9:269-273, Chen et al., 2002, J. Biol. Chem., 277:36521-36526).
It is presently unclear whether the differential cellular effects of PS1 mutations on Notch1 cleavage are attributed to cellular factors such as the maturation of the γ-secretase complex, trafficking of substrates or modulation of γ-secretase itself. Although a cell-free Notch/γ-secretase assay has been reported using N100Flag substrate (Moehlmann et al., 2002, Proc. Nat. Acad. Sci. USA, 99:8025-8030), it is a western analysis-based assay that is limited in its application for the characterization of γ-secretase since it is labor-intensive, has low throughput and is not quantitative. Moreover, it has been questioned whether N100Flag is processed by γ-secretase (Keller et al., 2006, Biochem., 45:5351-5358).